The method for transformation using Agrobacterium has a number of excellent features including, in general, the high efficiency, the small number of copies of the introduced gene, the feature that the gene may be introduced without fragmenting a specific region called T-DNA, and the feature that the frequency of mutation occurred during cultivation is low because transformants may be obtained by cultivation for a short period of time. Therefore, the method is widely used as the most useful method for transforming various plants.
Although the Agrobacterium method is an extremely excellent method for transforming plants, whether the transformation is successful or not and the transformation efficiency largely varies depending on the plant species, genotype and the plant tissue used (Potrykus et al. 1998 (Reference (33))). That is, there are species with which the transformation has not been successful, and species with which the transformation may be attained only with limited varieties. Further, there are species with which the tissue to be used is limited so that a large amount of materials cannot be treated. To prepare a practical variety by genetic recombination, it is necessary to prepare a large number of transformed plants and to select the line having the desired character therefrom. However, at present, the type of plants with which a large number of transformed plants may be prepared for this purpose is limited. Thus, to develop an improved method by which this problem may be overcome is strongly demanded.
Although the method for transformation via Agrobacterium differs in the starting material, composition of the culture medium and the like, it is almost common to the Agrobacterium method that the method comprises making a tissue which is a starting material contact a suspension of Agrobacterium, selecting transformed cells after co-culturing, and growing transformed plants. The Agrobacterium is infected without performing a special treatment except for sterilization treatment which is carried out as required (Rogers et al. 1988 (Reference (34)), Visser 1991 (Reference (38)), McCormick 1991 (Reference (29)), Lindsey et al. 1991 (Reference (28))). Thus, studies for improving transformation system has been carried out mainly on the Agrobacterium strain, constitution of the vector, composition of medium, types of selection marker gene and promoter, the type of the tissue used as the material, and the like.
On the other hand, studies for changing the plant tissue before infection of Agrobacterium to a physiological state in which the genes are likely to be introduced have been scarcely made. If the physiological state of the tissue can be changed to such a physiological state by a simple treatment, the method is very useful, and it is expected that, in addition to the promotion of the transformation efficiency, transformation may be attained for the species or genotypes with which transformation has been hitherto difficult, that is a prominent effect. Known studies about pretreatment of plant tissue include particle gun treatment (Bidney et al., 1992 (Reference (5))) and ultrasonication treatment (Trick et al., 1997 (Reference (37))). Both of these methods aim at promoting invasion of bacteria into the plant tissue by physically injuring the tissue, so as to increase the number of plant cells infected. However, these methods are nothing more than developments of the leaf disk method (Horsch et al., 1985 (Reference (17))) and not treatments based on novel concepts. The degree of effectiveness and universality of the methods have not been clarified, and they are not used as general methods.